Human Papillomavirus (HPV) is the most common sexually transmitted infectious agent worldwide and also is estimated to cause ˜500,000 cases of cancer a year (34). HPV infection is also associated with a variety of other diseases, including cutaneous and genital warts. Over 100 different HPV types have been identified, but the most common HPV-associated cancer, cervical cancer, is associated with infection by one of a subset of about 15-18 HPVs termed “high-risk” types. Two of the high-risk types, HPV16 and HPV18, account for approximately 70% of all cases of cervical cancer, and six other high-risk types (types 45, 31, 33, 52, 58, and 35) account for an additional 25% of all cases (33).
Conventional approaches for developing an HPV vaccine were not possible because of the lack of a tissue culture system for robust HPV propagation. However, in the early 1990s it was discovered that the L1 major capsid protein of HPV could self-assemble into virus-like particles (VLPs) that are structurally and antigenically similar to infectious virus (21, 24, 25, 43, 51). HPV VLPs are highly immunogenic; they induce high titer antibody responses upon vaccination of animals and people (44). These results paved the way for the development of the two commercially available STI HPV vaccines. Gardasil®, developed by Merck, contains L1-VLPs derived from two high-risk HPV types (16 and 18) and two low-risk HPVs associated with genital warts (6 and 11). Cervarix®, developed by GlaxoSmithKline, contains HPV16 and 18 L1-VLPs. These vaccines have excellent safety profiles, are highly effective at preventing infection and disease, and appear to induce long-lasting antibody responses (26).
Although some cross-reactivity has been observed between closely related HPV genotypes (46), the protection provided upon vaccination with L1-VLPs is largely HPV type-specific. For example, women who are vaccinated with HPV16 L1-VLPs are completely protected against HPV16-related disease (cervical intraepithelial neoplasia; CIN), but not against disease caused by other high-risk HPV types (26, 31). The type-specific nature of neutralizing antibodies induced by HPV16 L1-VLPs has also been confirmed using in vitro neutralization assays (35, 42). Taking into account the types covered by the current vaccine, and the possibility of some partial cross-protection, it is estimated that with complete vaccine coverage Gardasil® and Cervarix® could provide protection against ˜70-80% of cervical cancers. Yet, this figure may be an overestimate; ˜50% of infected women are infected with multiple carcinogenic types. Because infection with HPV16 and 18 can cause cancer more rapidly than other high-risk types, co-infection with HPV16 and HPV18 may mask the true risk of cancer caused by other HPV types. Because vaccinated populations are still at risk for cancer the American Cancer Society has recommended that vaccinated women continue to be subjected to Pap screening on an annual basis, at an annual cost of $4-5 billion in the United States alone.
Inducing Broadly Neutralizing Antibodies Against HPV by Targeting the Minor Capsid Protein, L2.
Papillomaviruses encode two capsid proteins, L1 and L2. Upon expression, the major capsid protein, L1, can spontaneously self-assemble into pentamers that further assemble into VLPs, comprised of 360 copies of L1. The minor capsid protein, L2, is not required for VLP formation, but is required for formation of infectious virions (and pseudovirions). Up to 72 copies of L2 can be incorporated in a VLP, and viral infectivity correlates with L2 content (5).
Although neither natural infection nor immunization with L1/L2 VLPs elicits anti-L2 antibody responses, vaccination with bacterially expressed L2 protein, or peptides derived from L2, results in the production of neutralizing antibodies that are protective in animal models (1, 7, 13, 19, 30). The somewhat contradictory data indicating that L2 is poorly immunogenic, yet is the target of highly neutralizing antibodies, can be explained by recent studies that shed light on the role of L2 during viral infection. Structural studies have shown that L2 is poorly exposed on the surface of virions (5). However, it has been proposed that after the virus binds to its primary cellular receptor the capsid undergoes a conformational change that exposes the amino terminus of L2 (15, 45). Once exposed, 12 amino acids at the N-terminus of L2 are cleaved by a cellular protein, furin, exposing one or more L2 neutralizing epitopes, and, it is theorized, allow virions to interact with a cellular coreceptor (15, 39).
Because L2 neutralizing epitopes are not exposed until after HPV binding, normal infection fails to induce anti-L2 neutralizing antibody responses. Thus, there has been little evolutionary pressure for L2 to undergo antigenic variation. Unlike L1-specific neutralizing antibodies, L2-specific neutralizing antibodies are broadly cross-neutralizing (41), suggesting that neutralizing epitopes on L2 are conserved across HPVs and even papillomaviruses (PVs) that infect different species. For example, antisera raised against a peptide representing amino acids 1-88 from bovine papillomavirus type 1 (BPV-1) L2 can cross-neutralize a diverse panel of mucosal and cutaneous HPVs (36). Similarly, vaccination with HPV16 and BPV-1 L2 peptides protects rabbits against challenge with two different rabbit papillomaviruses (19).
In principle, L2 vaccines may be able to overcome the high production cost and type-specific limitations of L 1-VLP vaccines. Unfortunately, however, the neutralizing titers produced upon L2 vaccination are considerably lower than for L1 VLP vaccines, particularly against heterologous HPV types (41). Therefore, it is likely that an L2 vaccine will only be effective if its immunogenicity is enhanced.
VLPs Induce Strong Antibody Responses.
Virus-like particles (VLPs) make excellent vaccines. They are non-infectious, often easier to produce than actual viruses, and, because the regularity of their capsid structure presents viral epitopes as dense, highly repetitive arrays that strongly stimulate B cells, they are highly immunogenic. VLPs are comprised of one or more proteins arranged geometrically into dense, repetitive arrays. These structures are largely unique to microbial antigens, and the mammalian immune system has apparently evolved to respond vigorously to this arrangement of antigens. B cells specifically recognize and respond strongly to the ordered array of densely spaced repetitive elements characteristic of virus surfaces (2, 18). Highly repetitive antigens provoke oligomerization of the membrane-associated immunoglobulin (Ig) molecules that constitute the B cell receptor (BCR) (3). There is evidence that the Ig crosslinking mediated by multivalent antigens leads to the formation of highly stable BCR-signaling microdomains that are associated with increased signaling to the B cell (48). This signaling stimulates B cell proliferation, migration, and upregulation of both major histocompatibility complex (MHC) class II and the co-stimulatory molecules that permit subsequent interactions with T helper cells that are required to trigger IgG secretion, affinity maturation, and the generation of long-lived memory B cells (9). Consequently, we and others have shown that multivalent antigens such as VLPs can activate B cells at much lower concentrations than monomeric antigens (4, 16, 17, 32). Hence, VLPs are innately immunogenic: they induce high titer and long lasting antibody responses at low doses, often without requiring adjuvants (22, 50).
VLPs as Flexible Platforms for Vaccine Development.
VLPs can be used as the basis for vaccines targeting the virus from which they were derived (the Hepatitis B virus vaccine and aforementioned HPV vaccine are two clinically approved VLP vaccines, other VLP vaccines are in clinical trials). However, they also can be used as platforms to display practically any epitope in a highly immunogenic, multivalent format. Heterologous antigens displayed at high density on the surface of VLPs exhibit the same high immunogenicity as unmodified VLPs. VLPs derived from a variety of different viruses have been exploited in this manner to induce antibody responses against heterologous targets that are poorly immunogenic in their native contexts. Although the VLP platform strategy has typically been applied to target antigens derived from pathogens, VLP-display can effectively induce antibody responses against practically any antigen. One example is the vaccine for nicotine addiction (designed to assist smokers who are trying to quit) developed by a biotechnology company, Cytos Biotechnology. This vaccine consists of nicotine, conjugated at high copy number to the surface of VLPs derived from a bacteriophage. In phase II clinical trials, VLPs displaying nicotine were well-tolerated and induced strong nicotine-specific IgG responses in 100% of immunized subjects (14). Even self-antigens, which are normally subject to the mechanisms of B cell tolerance, are immunogenic when displayed at high density on the surface of VLPs. Vaccines have been developed against self-molecules involved in several different diseases, including amyloid-beta (Alzheimers (12, 27)), TNF-α (arthritis (10)), CCR5 (HIV infection (8, 11)), gastrin (cancer, unpublished data), IgE (allergy, unpublished data), and others. VLP-based vaccines developed by pharmaceutical companies targeting amyloid-beta and angiotensin II (hypertension) are currently being evaluated in clinical trials; positive results from the trial of vaccine targeting angiotensin II (as a vaccine for hypertension) were reported in the spring of 2008 (49).
HPV vaccines that target the L2 protein have been described in a variety of other journal articles, patents, and patent applications.
For example, Kawana et al. describe a peptide representing amino acids 108-120 from HPV16 L2 that induces neutralizing antibodies effective against HPV16 and HPV11 (23). The information obtained from these results was said to be useful for developing a prophylactic peptide vaccine that prevents infection with genital HPVs in humans.
U.S. Pat. No. 6,174,532 and PCT/US2006/003601 described the use of the N-terminal portion of papillomavirus L2 protein or a prophylactically effective peptide fragment thereof (or a prophylactically effective peptide derivative sequence thereof) in the production of a medicament suitable for use as a prophylactic agent against papillomavirus infection in mammals.
U.S. Patent Application Document No. 2008/0213293 describes treating or preventing respiratory papillomatosis by immunizing either a mother or child before, during or after delivery. Immunity may be induced with a vaccine comprising a HPV peptide antigen fused to a viral protein or other antigen. Antibodies and cells may be recovered from an animal previously vaccinated with the same vaccine. Of particular interest is the use of HPV L2 peptides designating a neutralizing epitope of HPV.
PCT/US2008/053498 described the use of papillomavirus L2 polypeptides produced in a plant expression system as a prophylactic HPV vaccine.
PCT WO 93/00436 describes papillomavirus L2 protein for use in the production of a medicament for use in medicine, particularly for use in the prophylaxis or therapy of papillomavirus tumours.
PCT 2004/052395 describes a vaccine composition comprising an HPV L2 peptide in physical association with an HPV virus like particle (VLP).
PCT 2008/082719 describes a composition that includes: a papillomavirus virus-like particle including an L1 protein or polypeptide and a chimeric protein or polypeptide that contains at least a portion of an L2 protein 20 and a protein or polypeptide fragment including a first epitope; and a DNA molecule encoding a protein or polypeptide including a second epitope.